Bovine α-lactalbumin (BLA) was treated by ultrasonic at 150 W/cm for different times and subsequently glycated with mannose by dry-heating. Molecular weight, intrinsic fluorescence spectra, glycation sites and degree of modified BLA were observed. The proteinaceous high molecular weight components were formed after ultrasonic prior to glycated modification, while the conformational changes were obvious. Prior to ultrasonic pretreatment, K62, K114, and K122 of BLA were identified. After treated by ultrasound at 150 W/cm for 5, 10, 15, and 20 min, the sites were increased to four, four, five, and five, respectively. All glycated sites of modified BLA exhibited a higher degree of substitution per peptide (DSP) values compared to native BLA. Ultrasonic at 150 W/cm for 20 min revealed the most significant change in the BLA structure. Therefore, conformational changes, the intensified glycation site, and DSP value were responsible for the structural changes of BLA. Practical applications BLA is suitable as an ingredient for infant nutrition in food, and has immune-modulating, antioxidant, antibacterial, and antitumor activity etc. This study revealed that the structural changes of BLA induced by ultrasonic prior to glycated modification. It will be beneficial to understand the mechanism of the functional changes of modified BLA. Ultrasonic prior to glycated modification will be more likely to develop a practical technology to modify protein in the food industry, and improve the functional characteristics of food, such as produce hypo-allergenic cow's milk in future.